Pdx-1 or Pdx-1-VP16 protein transduction induces β-cell gene expression in liver-stem WB cells

Background Pancreatic duodenal homeobox-1 (Pdx-1) or Pdx-1-VP16 gene transfer has been shown to induce in vitro rat liver-stem WB cell conversion into pancreatic endocrine precursor cells. High glucose conditions were necessary for further differentiation into functional insulin-producing cells. Pdx-1 has the ability to permeate different cell types due to an inherent protein transduction domain (PTD). In this study, we evaluated liver-to-pancreas conversion of WB cells following Pdx-1 or Pdx-1-VP16 protein transduction. Findings WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. β-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into β-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and β-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein. Conclusion These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for β-cell engineering for diabetes cell therapy.


Background
The difficulties encountered in obtaining sufficient supply of transplantable β-cells is a major problem in cell therapy of type I diabetes. Liver may be a potential source of cells for β-cell engineering. Indeed, liver and pancreas derive from the same endodermal region during embryogenesis [1] and hepatocytes and β-cells share similar built-in glucose-sensing systems.
Among transcription factors involved in pancreatic β-cell specification, Pdx-1 plays a central role. All progenitors of the endocrine as well as the exocrine pancreas express Pdx-1 [2,3]. In the adult, Pdx-1 expression is mainly restrained to β-cells where it regulates important β-cell functions like insulin transcription. Several in vitro studies, using viral or stable plasmid gene transfer, show that Pdx-1 expression in hepatic cells results in reprogrammation into insulin producing cells [4][5][6][7]. Fusion of Pdx-1 to the VP16 activation domain from Herpes simplex virus (Pdx-1-VP16) leads to more efficient liver-to-pancreas conversion than Pdx-1 alone [8][9][10][11][12]. Stable mouse Pdx-1 or Pdx-1-VP16 gene transfection initiates conversion of rat epithelial liver stem-like WB cell line into pancreatic endocrine precursor cells [13,14]. In these cells, long-term high glucose (HG) culture in vitro is necessary for further pancreatic endocrine differentiation.
Safety of gene therapy remaining a prime concern, protein transduction offers a more secure alternative to induce stem cell differentiation. Indeed, protein transduction domains (PTD) allow proteins to translocate across the cytoplasmic membrane. Due to an Antennapedia-like PTD in its structure, Pdx-1 protein can permeate different cell types and induces insulin expression in pancreatic ducts [15,16]. However, in human embryonic stem cells, the adjunction of a PTD domain derived from the HIV TAT protein (TAT) is necessary for Pdx-1 cell transduction [17]. Up to now, no study using protein transduction has achieved liver-to-endocrine pancreas conversion in vitro.
Here, we evaluate if Pdx-1(-VP16) proteins have the capacity to induce a pancreatic endocrine shift in WB cells. To achieve this, we treat WB cells with Pdx-1 or Pdx-1-VP16 proteins, containing their own PTD, or fused to the PTD of TAT.
To evaluate transduction efficiency, 50 × 10 3 WB cells/well were seeded onto a 24-well plate. 24 h later, the medium was replaced with fresh medium containing 15 μM eGFP, PTD Pdx-1 -eGFP, or TAT-eGFP proteins and incubated for another 24 h before confocal microscopy analysis.

Pdx-1 and TAT PTDs transduced HepG2 and WB cells
To assess PTD Pdx-1 -or TAT-mediated protein transduction in liver cells in vitro, we evaluated the ability of Pdx-1 and TAT PTDs to deliver fusion proteins into WB and HepG2 cells. To achieve this, we produced three recombinant eGFP fusion proteins: PTD Pdx-1 -eGFP, TAT-eGFP and eGFP alone as a negative control (Fig. 1a). Intracellular eGFP fluorescence was detected in cells treated with PTD Pdx-1 -eGFP or TAT-eGFP -as punctuated or diffused signals (Fig. 1b). No cellular uptake was detected in cells treated with eGFP protein lacking PTD (Fig. 1b). These results indicated clearly (i) that the efficiency of HepG2 and WB cell transduction was dependant on the presence of a PTD domain, and (ii) that PTD Pdx-1 efficiently delivered fusion proteins into cells.

Pdx-1(-VP16) protein transduction increased insulin promoter activity
In order to convert WB cells into insulin producing, cells we produced and purified four Pdx-1 proteins: Pdx-1, Pdx-1-VP16, TAT-Pdx-1 and TAT-Pdx-1-VP16 (Fig. 2a). Rat insulin-II promoter activity was significantly increased by treatment with any of the Pdx-1(-VP16) proteins (Fig. 2b). These results showed that these four Pdx-1(-VP16) proteins transduced hepatic cells and, in part, went to the nucleus and activated a β-cell specific promoter. Fusion of TAT to these proteins, which consequently contain two PTDs in their structure, increased insulin promoter activity in an equal manner as PTD Pdx-1 alone. Pdx-1 fusion to VP16 activation domain did not further enhance insulin promoter activity.

Pdx-1, Pdx-1-VP16, TAT-Pdx-1 and TAT-Pdx-1-VP16 proteins induced the expression of insulin and pancreaticrelated genes in WB cells
Whereas LG control cells expressed amylase and weakly insulin 2, but none of the other pancreatic genes tested here, HG control cells expressed transcription factors implicated in endocrine pancreatic differentiation: Ngn3, Nkx2.2 and Pdx-1 (Fig. 3). Expression of Kir6.2 was also detected. See additional data 3 for original electrophoresis gel images.
Taking advantage of the commitment of WB cells towards an endocrine phenotype in HG culture, Pdx-1(-VP16) protein treatment was performed on WB cells grown in HG medium. Proteins induced the expression of a wide panel of β-cell genes after two weeks of culture: NeuroD, Pax4, Nkx6.1, insulin 1, Glut-2. Expression of Pancreatic Polypeptide (PP), a non-β endocrine hormone, was also detected. Among different samples, some expressed all of these β-cell genes (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1,  Pdx-1, insulin 1, Glut-2, Kir6.2) and others only some of them. Although insulin 1 was expressed, insulin 2 expression was not induced by Pdx-1(-VP16) protein treatment. Glucokinase (GK) was not detected despite using primers amplifying pancreatic as well as hepatic GK isoforms.
No pancreatic endocrine gene expression was observed at earlier time points (day 6 and 9 of treatment, data not shown).
The hepatic marker Albumin was absent in all conditions tested, including LG control cells.

Discussion
In the present study, we demonstrate the aptitude of Pdx-1 or Pdx-1-VP16 protein transduction to reprogram hepatic stem-like cells into β-like cells in vitro.
First, we verified that the PTD of Pdx-1 and TAT allow transduction into hepatic WB and HepG2 cell lines. Intracellular localization of fusion proteins revealed by confocal microscopy analysis indicate a true uptake of proteins and not mere adherence to the cell surface. Furthermore, transduced recombinant Pdx-1, TAT-Pdx-1, Pdx-1-VP16 and TAT-Pdx-1-VP16 proteins exert biological activity on an insulin promoter reporter system. Pdx-1 fusion to VP16 activation domain does not further enhance insulin promoter activity consistent with previous findings [10]. Our results confirm reports of Pdx-1 transduction [15,20] and are the first demonstration of Pdx-1-VP16 protein transduction.
Pancreatic differentiation experiments were conducted in an HG environment. In fact, long-term HG culture furthers liver cell commitment towards a pancreatic fate [4,6,13,14,21]. Previous studies of Pdx-1(-VP16) expression in WB cells do not distinguish between respective contributions of transgene expression and HG culture on differentiation. Here, we show that HG culture alone converts WB cells into pancreatic endocrine precursor cells. In contrast to LG cultures, HG cultures express four pancreatic endocrine genes: Ngn3, Nkx2.2, Pdx-1 and Kir6.2. These results concur with Yang et al. study, where confluent culture of hepatic oval stem cells for 2 months in HG medium induces conversion into insulin-producing cells [21]. Kir6.2 expression is at odds with previous reports where Kir6.2 was detected in WB cells overexpressing Pdx-1 or Pdx-1-VP16 genes only (i) after 2-3 months in HG culture [14], (ii) 40 days post-transplantation into diabetic mouse [13], or (iii) after Pax4 co-expression [22]. Treated cells were observed by confocal microscopy without being fixed in order to exclude artifactual protein uptake [24]. CM-Dil was used to visualize cytoplasmic membrane (red staining). Scale bars = 50 μm.
Pdx-1, TAT-Pdx-1, Pdx-1-VP16, or TAT-Pdx-1-VP16 proteins further induce expression of NeuroD, Pax4, Nkx6.1, insulin 1 and Glut-2 after two weeks of treatment. Some samples express just a part of these markers or display RT-PCR expression patterns similar to HG controls. Quantitative analysis would help to further nuance pancreatic gene expression in-between these samples, in particular up-regulation of the endogenous Pdx-1 gene. The heterogeneity between samples may be a consequence of: (i) infrequent liver to endocrine pancreas conversion, leading to few pancreatic gene positive cells which may be difficult to detect, (ii) different kinetics of gene expression between wells. According to Tang et al. study, our experiments do not reveal more efficient differentiation following Pdx-1-VP16 protein treatment compared to Pdx-1 protein treatment [14]. TAT-mediated transduction does not lead to more advanced differentiation suggesting that containing two PTDs (PTD Pdx-1 and TAT) does not increase transduction efficiency. Surprisingly, insulin 1 and Pax4, two of the pancreatic genes expressed after our protein treatments, are not detected in a previous in vitro study on WB cells transduced with Pdx-1 or Pdx-1-VP16 genes, even after 3 months of HG culture [14]. Here, despite insulin 1 expression, neither insulin 2 nor glucokinase are detected pointing at the possible need for long-term culture in HG medium to obtain mature β-like cells [14]. Moreover, Ngn3 expression in wells scoring positive for all other pancreatic genes, including insulin 1, suggests the presence of remaining subpopulations of immature pancreatic precursor cells.
Gene expression profile of WB-cells treated with Pdx-1(-VP16) or TAT-Pdx-1(-VP16) proteins in high glucose culture Figure 3 Gene expression profile of WB-cells treated with Pdx-1(-VP16) or TAT-Pdx-1(-VP16) proteins in high glucose culture. RT-PCR analysis was performed after two weeks of protein treatment. cDNA from rat pancreas served as control for pancreatic gene expression, and cDNA from rat liver served as positive control for albumin expression (*). Each sample is represented by a single column and corresponds to an independent biological repeat of the experiment (LG (n = 5), HG (n = 5), Pdx-1 (n = 4), TAT-Pdx-1 (n = 4), Pdx-1-VP16 (n = 5), TAT-Pdx-1-VP16 (n = 5)). Each gene is represented by a single row of coloured boxes. Black colouring represents RT-PCR positive samples, whereas white colouring represents negative samples.
LG: Low Glucose control WB cells; HG: High Glucose control WB cells. (See additional data 3 for original electrophoresis gel images). expressed in rat liver or exocrine pancreatic cells, is detected in all conditions tested here [23].
Recently, Koya et al. demonstrated that Pdx-1 protein delivery into diabetic mice restores euglycemia mainly through pancreatic β-cell regeneration. The authors observed β-cell gene expression and insulin synthesis in the pancreas and in the liver of treated mice suggesting hepatic insulin contribution to euglycemia [20]. In complement, our findings provide the first direct evidence that Pdx-1(-VP16) protein transduction in conjunction with HG culture reprograms hepatic stem-like cells into cells displaying similarities with β-cells in vitro. At a point where strategies for targeted β-cell differentiation begin to surface, our study illustrates how simple exposition to Pdx-1(-VP16) protein in the surrounding medium triggers short-term pancreatic endocrine conversion. This study may contribute to the development of protein transduction therapy, a new concept to induce β-cell differentiation.